Identification of protein complexes required for efficient sister chromatid cohesion

Ctf8p is a component of Ctf18-RFC, an alternative replication factor C-like complex required for efficient sister chromatid cohesion in Saccharomyces cerevisiae. We performed synthetic genetic array (SGA) analysis with a ctf8 deletion strain as a primary screen to identify other nonessential genes required for efficient sister chromatid cohesion. We then assessed proficiency of cohesion at three chromosomal loci in strains containing deletions of the genes identified in the ctf8 SGA screen. Deletion of seven genes (CHL1, CSM3, BIM1, KAR3, TOF1, CTF4, and VIK1) resulted in defective sister chromatid cohesion. Mass spectrometric analysis of immunoprecipitated complexes identified a physical association between Kar3p and Vik1p and an interaction between Csm3p and Tof1p that we confirmed by coimmunoprecipitation from cell extracts. These data indicate that synthetic genetic array analysis coupled with specific secondary screens can effectively identify protein complexes functionally related to a reference gene. Furthermore, we find that genes involved in mitotic spindle integrity and positioning have a previously unrecognized role in sister chromatid cohesion.     

The mechanism of sister chromatid cohesion

Each of our cells inherit their genetic information in the form of chromosomes from a mother cell. In order that we obtain the full genetic complement, cells need to ensure that replicated chromosomes are accurately split and distributed during cell division. Mistakes in this process lead to aneuploidies, cells with supernumerous or missing chromosomes. Most aneuploid human embryos are not viable, and if they are, they develop severe birth defects. Aneuploidies later in human life are frequently found associated with the development of malignant cancer. DNA replication during S-phase is linked to segregation of the sister copies in mitosis by sister chromatid cohesion. A chromosomal protein complex, cohesin, holds replicated sister DNA strands together after their synthesis. This allows pairs of replication products to be recognised by the spindle apparatus in mitosis for segregation into opposite direction. At anaphase onset, cohesin is destroyed by a site-specific protease, separase. Here I review what we have learned about the molecular mechanism of sister chromatid cohesion. Cohesin forms a large proteinaceous ring that may hold sister chromatids by encircling and topological trapping. To understand how cohesin links newly synthesised replication products, biochemical assays to study the enzymology of cohesin will be required.     

Two putative acetyltransferases, san and deco, are required for establishing sister chromatid cohesion in Drosophila

BACKGROUND: Sister chromatid cohesion is needed for proper alignment and segregation of chromosomes during cell division. Chromatids are linked by the multiprotein cohesin complex, which binds to DNA during G(1) and then establishes cohesion during S phase DNA replication. However, many aspects of the mechanisms that establish and maintain cohesion during mitosis remain unclear.RESULTS: We found that mutations in two evolutionarily conserved Drosophila genes, san (separation anxiety) and deco (Drosophila eco1), disrupt centromeric sister chromatid cohesion very early in division. This failure of sister chromatid cohesion does not require separase and is correlated with a failure of the cohesin component Scc1 to accumulate in centromeric regions. It thus appears that these mutations interfere with the establishment of centromeric sister chromatid cohesion. Secondary consequences of these mutations include activation of the spindle checkpoint, causing metaphase delay or arrest. Some cells eventually escape the block but incur many errors in anaphase chromosome segregation. Both san and deco are predicted to encode acetyltransferases, which transfer acetyl groups either to internal lysine residues or to the N terminus of other proteins. The San protein is itself acetylated, and it associates with the Nat1 and Ard1 subunits of the NatA acetyltransferase.CONCLUSIONS: At least two diverse acetyltransferases play vital roles in regulating sister chromatid cohesion during Drosophila mitosis.     

Calpain 2 is required for sister chromatid cohesion

Calpains form a family of Ca²⁺-dependent cysteine proteases involved in diverse cellular processes. However, the specific functions of each calpain isoform remain unknown. Recent reports have shown that calpain 2 (Capn2) is essential for cell viability. We have recently shown that Capn2 is a nuclear protease associated with chromosomes during mitosis in mammalian embryonic cells. We now report that Capn2 depletion impairs mitosis and induces apoptosis in murine cells. Low Capn2 levels induce chromosome alignment defects, the loss of histone H3 threonine 3 phosphorylation at centromeres, and premature sister chromatid separation. Thus, Capn2 may play a role in fundamental mitotic functions, such as the maintenance of sister chromatid cohesion.     

The sister-chromatid cohesion protein ORD is required for chiasma maintenance in Drosophila oocytes

Accurate chromosome partitioning during cell division requires that cohesion hold sister chromatids together until kinetochores correctly attach to spindle microtubules. In 1932, Darlington noted that sister-chromatid cohesion distal to the site of exchange also could play a vital role in maintaining the association of chiasmate homologs during meiosis. Cohesion linking a recombinant chromatid with a sister of each homologous pair would resist spindle forces that separate kinetochores of homologous chromosomes (see Figure 1). Although centromeric cohesion must be retained to ensure proper segregation during meiosis II, dissolution of arm cohesion would be required for anaphase I to occur. This hypothesis is supported by recent evidence in yeast and C. elegans that separase activity is essential for the segregation of recombinant homologs during meiosis I. We present evidence that Drosophila oocytes require sister-chromatid cohesion to maintain a physical attachment between recombinant chromosomes. Using FISH to monitor cohesion directly, we confirm that oocytes lacking ORD activity exhibit cohesion defects, consistent with previous genetic results. We also show that ord(null) oocytes that have undergone recombination are unable to arrest at metaphase I, indicating that chiasmata are unstable in the absence of cohesion. Our results support the model that arm cohesion provides a conserved mechanism that ensures physical attachment between recombinant homologs until anaphase I.     

Targeted sister chromatid cohesion by Sir2

The protein complex known as cohesin binds pericentric regions and other sites of eukaryotic genomes to mediate cohesion of sister chromatids. In budding yeast Saccharomyces cerevisiae, cohesin also binds silent chromatin, a repressive chromatin structure that functionally resembles heterochromatin of higher eukaryotes. We developed a protein-targeting assay to investigate the mechanistic basis for cohesion of silent chromatin domains. Individual silencing factors were tethered to sites where pairing of sister chromatids could be evaluated by fluorescence microscopy. We report that the evolutionarily conserved Sir2 histone deacetylase, an essential silent chromatin component, was both necessary and sufficient for cohesion. The cohesin genes were required, but the Sir2 deacetylase activity and other silencing factors were not. Binding of cohesin to silent chromatin was achieved with a small carboxyl terminal fragment of Sir2. Taken together, these data define a unique role for Sir2 in cohesion of silent chromatin that is distinct from the enzyme's role as a histone deacetylase.     

A physical assay for sister chromatid cohesion in vitro

Cohesion between sister chromatids depends on a multiprotein complex called cohesin that has been proposed to hold sister DNAs together by trapping them inside a large tripartite ring. Sister chromatid cohesion has hitherto only been detected by using cytological methods in living cells. We show here that cohesion between the sister DNAs of circular minichromosomes established in vivo can be detected in vitro by velocity gradient sedimentation and agarose-gel electrophoresis. This ex vivo cohesion does not depend on intercatenation of sister DNAs but is destroyed by cleavage of cohesin's Scc1 subunit or minichromosome linearization. These data represent the best evidence so far that the cohesin ring physically holds sister DNAs together and are consistent with the notion that it does so by using a topological principle involving the trapping DNAs inside its ring.     

Regulation of sister chromatid cohesion between chromosome arms

Sister chromatid separation in anaphase depends on the removal of cohesin complexes from chromosomes. In vertebrates, the bulk of cohesin is already removed from chromosome arms during prophase and prometaphase, whereas cohesin remains at centromeres until metaphase, when cohesin is cleaved by the protease separase. In unperturbed mitoses, arm cohesion nevertheless persists throughout metaphase and is principally sufficient to maintain sister chromatid cohesion. How arm cohesion is maintained until metaphase is unknown. Here we show that small amounts of cohesin can be detected in the interchromatid region of metaphase chromosome arms. If prometaphase is prolonged by treatment of cells with microtubule poisons, these cohesin complexes dissociate from chromosome arms, and arm cohesion is dissolved. If cohesin dissociation in prometaphase-arrested cells is prevented by depletion of Plk1 or inhibition of Aurora B, arm cohesion is maintained. These observations imply that, in unperturbed mitoses, small amounts of cohesin maintain arm cohesion until metaphase. When cells lacking Plk1 and Aurora B activity enter anaphase, chromatids lose cohesin. This loss is prevented by proteasome inhibitors, implying that it depends on separase activation. Separase may therefore be able to cleave cohesin at centromeres and on chromosome arms.     

Reduced sister chromatid cohesion acts as a tumor penetrance modifier

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.     

A multi-step pathway for the establishment of sister chromatid cohesion

The cohesion of sister chromatids is mediated by cohesin, a protein complex containing members of the structural maintenance of chromosome (Smc) family. How cohesins tether sister chromatids is not yet understood. Here, we mutate SMC1, the gene encoding a cohesin subunit of budding yeast, by random insertion dominant negative mutagenesis to generate alleles that are highly informative for cohesin assembly and function. Cohesins mutated in the Hinge or Loop1 regions of Smc1 bind chromatin by a mechanism similar to wild-type cohesin, but fail to enrich at cohesin-associated regions (CARs) and pericentric regions. Hence, the Hinge and Loop1 regions of Smc1 are essential for the specific chromatin binding of cohesin. This specific binding and a subsequent Ctf7/Eco1-dependent step are both required for the establishment of cohesion. We propose that a cohesin or cohesin oligomer tethers the sister chromatids through two chromatin-binding events that are regulated spatially by CAR binding and temporally by Ctf7 activation, to ensure cohesins crosslink only sister chromatids.     

S-phase checkpoint genes safeguard high-fidelity sister chromatid cohesion

Cohesion establishment and maintenance are carried out by proteins that modify the activity of Cohesin, an essential complex that holds sister chromatids together. Constituents of the replication fork, such as the DNA polymerase alpha-binding protein Ctf4, contribute to cohesion in ways that are poorly understood. To identify additional cohesion components, we analyzed a ctf4Delta synthetic lethal screen performed on microarrays. We focused on a subset of ctf4Delta-interacting genes with genetic instability of their own. Our analyses revealed that 17 previously studied genes are also necessary for the maintenance of robust association of sisters in metaphase. Among these were subunits of the MRX complex, which forms a molecular structure similar to Cohesin. Further investigation indicated that the MRX complex did not contribute to metaphase cohesion independent of Cohesin, although an additional role may be contributed by XRS2. In general, results from the screen indicated a sister chromatid cohesion role for a specific subset of genes that function in DNA replication and repair. This subset is particularly enriched for genes that support the S-phase checkpoint. We suggest that these genes promote and protect a chromatin environment conducive to robust cohesion.     

Sororin mediates sister chromatid cohesion by antagonizing Wapl

Sister chromatid cohesion is essential for chromosome segregation and is mediated by cohesin bound to DNA. Cohesin-DNA interactions can be reversed by the cohesion-associated protein Wapl, whereas a stably DNA-bound form of cohesin is thought to mediate cohesion. In vertebrates, Sororin is essential for cohesion and stable cohesin-DNA interactions, but how Sororin performs these functions is unknown. We show that DNA replication and cohesin acetylation promote binding of Sororin to cohesin, and that Sororin displaces Wapl from its binding partner Pds5. In the absence of Wapl, Sororin becomes dispensable for cohesion. We propose that Sororin maintains cohesion by inhibiting Wapl's ability to dissociate cohesin from DNA. Sororin has only been identified in vertebrates, but we show that many invertebrate species contain Sororin-related proteins, and that one of these, Dalmatian, is essential for cohesion in Drosophila. The mechanism we describe here may therefore be widely conserved among different species.     

Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

Sororin is a master regulator of sister chromatid cohesion and separation

The maintenance of sister chromatid cohesion from S phase to the onset of anaphase relies on a small but evolutionarily conserved protein called Sororin. Sororin is a phosphoprotein and its dynamic localization and function are regulated by protein kinases, such as Cdk1/cyclin B and Erk2. The association of Sororin with chromatin requires cohesin to be preloaded to chromatin and modification of Smc3 during DNA replication. Sororin antagonizes the function of Wapl in cohesin releasing from S to G₂ phase and promotes cohesin release from sister chromatid arms in prophase via interaction with Plk1. This review focuses on progress of the identification and regulation of Sororin during cell cycle; role of post-translational modification on Sororin function; role of Sororin in the maintenance and resolution of sister chromatid cohesion; and finally discusses Sororin’s emerging role in cancer and the potential issues that need be addressed in the future.     

Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.